Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Genera Burkholderia and Lipomyces are predominant aluminum-resistant microorganisms isolated from acidic forest soils using cycloheximide-amended growth media

Genera Burkholderia and Lipomyces are predominant aluminum-resistant microorganisms isolated from... Ann Microbiol (2012) 62:1339–1344 DOI 10.1007/s13213-011-0393-4 SHORT COMMUNICATION Genera Burkholderia and Lipomyces are predominant aluminum-resistant microorganisms isolated from acidic forest soils using cycloheximide-amended growth media Takashi Kunito & Miki Owaki & Yasutaka Ihyo & Hirotaka Sumi & Hideshige Toda & Daisuke Fukuda & Ho-Dong Park Received: 20 September 2011 /Accepted: 15 November 2011 /Published online: 29 November 2011 # Springer-Verlag and the University of Milan 2011 . . . Abstract In acidic forest soils, microorganisms should Keywords Aluminum Burkholderia Forest soil adapt to toxicity of aluminum (Al), which is solubilized by Lipomyces Resistance acidity. We hypothesized that Al-resistant bacteria are diverse, especially in soils with high levels of Al, because these bacteria are expected to have adapted to Al stress over a Introduction long time. We isolated Al-resistant bacteria from acid forest soils using diluted tryptic soy broth agar plates with added Al Aluminum has a higher affinity for phosphorus (P) and and cycloheximide, and examined the relationship between slower reaction kinetics than calcium (Ca) or magnesium their diversity and Al levels in soils. Based on 16S rDNA (Mg), and is thus a powerful inhibitor of many biological sequences, 10 out of 11 isolated bacteria were assigned to the processes dependent on Ca and Mg (Macdonald and Martin genus Burkholderia, and 1 to the genus Acinetobacter. 1988; Exley and Birchall 1992). Aluminum toxicity is a Although cycloheximide was added to the Al-enriched agar, major problem in acid soils because Al solubility increases yeasts were isolated from soils, and were examined. On the with decreasing soil pH (Sposito 1996). Acidic precipitation basis of ITS1, 5.8S rDNA, and ITS2 sequences, 13 out of 14 has therefore exaggerated the adverse effects of Al on plants yeast isolates were assigned to Lipomyces sp. and 1 isolate and microorganisms in these soils. High levels of Al in soils to Cryptococcus sp. Diversity of Al-resistant bacteria was may cause root damage and mineral imbalances in trees, and low in acidic forest soils, and was not related to Al levels in even result in forest decline (Godbold et al. 1988). soils. Population numbers of Al-resistant microorganisms, Microorganisms are known to be more sensitive to Al tox- however, increased with increasing Al levels. icity than are trees (Joner et al. 2005). However, few studies have assessed the adverse effects of Al on soil microorgan- isms or their adaptation to Al stress (Piña and Cervantes 1996). In contrast, the environmental behavior of Al in soils : : : : : T. Kunito (*) M. Owaki Y. Ihyo H. Sumi H. Toda (Sposito 1996), and the adaptation of plants to Al toxicity H.-D. Park Department of Environmental Sciences, Faculty of Science, (Matsumoto 2000), have been well studied. Aluminum Shinshu University, directly affects microorganisms through its fixation to cell 3-1-1 Asahi, walls, fixation to DNA via binding with P, and its toxic Matsumoto 390-8621, Japan effects on enzymes (Robert 1995). It indirectly affects e-mail: kunito@shinshu-u.ac.jp microorganisms through perturbation of Ca and Mg metab- D. Fukuda olism (Robert 1995), leading to reductions in microbial Daiichi Sankyo Co. Ltd, biomass, basal respiration, ATP levels, and protease activity 1-2-58 Hiromachi, in soils (Illmer et al. 2003). Al-resistant microorganisms are Shinagawa-ku, Tokyo 140-8710, Japan 1340 Ann Microbiol (2012) 62:1339–1344 therefore likely to play a key role in various microbially- soils were classified as Haplic Brown Forest soils mediated processes such as nutrient cycling in soils. (Inceptisols). Vegetation was predominately larch, Larix Bacterial Al resistance systems seem to be unspecific and kaempferi, in five sites (A1, A3, B1, B2, and B3), passive (meaning that systems have another primary role, Japanese red pine, Pinus densiflora, in one site (A2), such as production of extracellular polysaccharides; Robert Japanese chestnut, Castanea crenata, and Mongolian oak, 1995). Bacteria have, however, developed sophisticated re- Quercus mongolica, in one site (B4), and Veitch fir, Abies sistance mechanisms to various heavy metals, including veitchii, and Maries fir, Abies mariesii, in one site (B5). efflux systems for cadmium (Cd), lead (Pb), copper (Cu), Sampling sites were located at altitudes between 480 and and zinc (Zn), and volatilization of mercury (Hg) (Silver and 2,050 m. The mean annual precipitation at meteorological Phung 2005). No specific genes for Al resistance have so far stations adjacent to sampling sites ranged from 941 to been found in bacteria (Silver and Phung 2005). The most 2,152 mm, and the air temperature from 7.1 to 16.5°C. extensively investigated is Pseudomonas fluorescens,in Each soil sample was sieved through a 2-mm mesh and which Al is secreted in association with oxalate and phos- homogenized well. A portion of each sample was air-dried for phatidylethanolamine by an energy-independent process chemical analysis, while the remainder was kept field-moist at (neither proton-motive force nor ATP hydrolysis), and is 4°C. Soil properties were determined using standard methods immobilized in the insoluble gelatinous precipitate (Hamel as described elsewhere (Kunito et al. 2009, 2011); these are and Appanna 2003; Lemire et al. 2010). shown in Table 1. Exchangeable Al, a fraction toxic to Acidic forest soils with high exchangeable Al concentra- microbes (Illmer et al. 1995) and plants (Saigusa et al. tions are widely distributed in Japan, and have developed 1980), was extracted using 1 M KCl, and analyzed using through natural processes across long periods of time. We an atomic absorption spectrometer 5100ZL (Perkin Elmer, hypothesized that Al-resistant bacteria are diverse, especially Tokyo). All data are expressed on a dry weight basis. in soils with high levels of Al, because these bacteria are expected to have adapted to Al stress in acidic forest soils over Isolation and identification of Al-resistant microorganisms a long time. To examine this, we isolated and identified pre- dominant Al-resistant bacteria from Japanese acidic forest soils. Moist soil samples were dispersed in sterile tap water using a Waring blender 500 C (Sakuma, Tokyo), and the resulting slurry was decimally diluted with sterile tap water. Samples Materials and methods were spread on a 15-fold-diluted TSB agar plate (tryptic soy broth, 2 g; agar, 10 g; cycloheximide, 50 mg; distilled water, Soils 1 L; pH 4.0), with added Al at 1.5 mM. The Al level in the medium selected about 10% of the microbial population Soil samples were collected from A horizons in forests in growing on a 15-fold-diluted TSB agar plate (pH 4.0) with- Nagano Prefecture, Japan. Three soils were classified as out Al. For preparation of the diluted TSB agar plate, an allophanic Kuroboku soils (allophanic Andisols), and five acidified TSB medium with HCl, an AlCl solution, and an Table 1 Properties of forest soils used Soil no. Latitude Longitude Altitude Soil order Soil group (Japan) pH Organic Total N Exchangeable −1 (N) (E) (m) (USDA) C (%) (%) Al (mg kg ) A1 36°24′ 138°03′ 1,510 Andisol Allophanic 6.1 5.2 0.35 1.06 Kuroboku soil A2 35°55′ 138°02′ 880 Andisol Allophanic 5.2 8.2 0.69 56.7 Kuroboku soil A3 35°50′ 137°52′ 1,300 Andisol Allophanic 4.9 8.6 0.54 145 Kuroboku soil B1 36°31′ 138°10′ 480 Inceptisol Haplic Brown 6.9 2.9 0.23 0.45 Forest soil B2 35°51′ 137°50′ 1,560 Inceptisol Haplic Brown 4.9 8.8 0.69 104 Forest soil B3 35°50′ 137°50′ 1,870 Inceptisol Haplic Brown 4.4 10.2 0.69 146 Forest soil B4 36°26′ 138°20′ 1,100 Inceptisol Haplic Brown 4.5 18.0 1.0 217 Forest soil B5 35°50′ 137°49′ 2,050 Inceptisol Haplic Brown 4.1 21.9 1.1 436 Forest soil Ann Microbiol (2012) 62:1339–1344 1341 agar solution were separately autoclaved to prevent hydro- solution, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% lysis of the agar (Kanazawa and Kunito 1996). After cooling Triton X-100, 1.5 mM MgCl , 0.2 mM of each dNTP, and to 50ºC, these three solutions were mixed, and filter- 1 U of ExTaq polymerase (Takara Bioscience, Tokyo). sterilized cycloheximide was added. The pH of the resulting Amplification was carried out over 30 cycles (denaturation agar plate was confirmed as 4.0 in a preliminary experiment. at 95ºC for 30 s, annealing at 55ºC for 30 s, and extension at After 10 days incubation at 25ºC, five colonies were ran- 72ºC for 1 min). Amplified fragments of 16S rDNA were domly isolated from each soil sample, and subjected to purified using ExoSAP-IT (GE Healthcare Bioscience, single colony isolation. Single colony isolation, however, Tokyo), and were sequenced directly. Although cyclohexi- was only partially successful (Table 2) because of obstruc- mide, an inhibitor of cytosolic protein biosynthesis in eukar- tion by fungal growth on the plate, despite the addition of yotes, was added to the Al-enriched agar, yeasts and not cycloheximide. bacteria were isolated from four soils (Table 2). These iso- Genomic DNA of isolated bacteria was extracted lates were identified based on ITS1, 5.8S rDNA, and ITS2 according to Tago et al. (2006), and the isolates sequences. These were amplified using primers ITS1-F were identified based on 16S rRNA gene sequences. These (Gardes and Bruns 1993) and ITS4 (White et al. 1990) after were amplified by PCR using universal primers; 27f extraction of nuclear DNA by the method of Makimura et al. (5′-AGAGTTTGATCATGGCTCAG-3′) and 1492r (5′- (1994). The PCR protocol and sequence analysis were as GGCTACCTTGTTACGACTT-3′). PCR was performed in described above. Phylogenetic analysis was performed using 10 μl of a mixture containing 1 μl of genomic DNA CLUSTAL X version 2 (Larkin et al. 2007). Table 2 Characteristics of aluminum-resistant bacteria and yeasts from forest soils Isolates Soil no. Accession no. Tentative identification Doubling time (hr) Ratio of doubling time (Diluted TSB+1 mM Al) / (Diluted TSB) Diluted TSB Diluted TSB+1 mM Al Bacteria BA1a A1 AB665296 Acinetobacter sp. 7.20 7.47 1.04 BA3a A3 AB665286 Burkholderia sp. 3.31 4.19 1.27 BB4a B4 AB665287 Burkholderia sp. 2.77 3.48 1.26 BB4b B4 AB665288 Burkholderia sp. 2.86 3.12 1.09 BB4c B4 AB665289 Burkholderia sp. 3.15 4.89 1.55 BB4d B4 AB665290 Burkholderia sp. 3.30 5.56 1.68 BB4e B4 AB665291 Burkholderia sp. 3.14 6.33 2.02 BB5a B5 AB665292 Burkholderia sp. 3.35 3.51 1.05 BB5c B5 AB665293 Burkholderia sp. 3.04 3.31 1.09 BB5d B5 AB665294 Burkholderia sp. 2.73 3.35 1.23 BB5e B5 AB665295 Burkholderia sp. 2.69 3.08 1.14 Yeasts YA2a A2 AB665297 Lipomyces sp. 7.99 7.62 0.95 YA2b A2 AB665310 Cryptococcus sp. 4.67 4.23 0.91 YA2c A2 AB665298 Lipomyces sp. 6.42 7.14 1.11 YA2d A2 AB665299 Lipomyces sp. 6.01 6.46 1.07 YA2e A2 AB665300 Lipomyces sp. 5.33 5.45 1.02 YB1a B1 AB665301 Lipomyces sp. 6.83 6.51 0.95 YB1b B1 AB665302 Lipomyces sp. 6.92 7.49 1.08 YB2a B2 AB665303 Lipomyces sp. 6.45 6.32 0.98 YB2b B2 AB665304 Lipomyces sp. 6.03 6.19 1.03 YB2c B2 AB665305 Lipomyces sp. 6.42 6.56 1.02 YB2d B2 AB665306 Lipomyces sp. 6.20 5.83 0.94 YB2e B2 AB665307 Lipomyces sp. 6.26 6.22 0.99 YB3a B3 AB665308 Lipomyces sp. 6.17 6.99 1.13 YB3b B3 AB665309 Lipomyces sp. 5.02 7.17 1.43 TSB, tryptic soy broth 1342 Ann Microbiol (2012) 62:1339–1344 Evaluation of Al resistance of isolates The numbers of colonies on diluted TSB agar plates with 4 6 1.5 mM Al added ranged from 3.9×10 to 4.6×10 per gram Levels of Al resistance were evaluated using a ratio of of dry soil, with a significant positive correlation between doubling time in 1.5-fold-diluted TSB (pH 4.0) amended with the number of Al-resistant colonies and the exchangeable Al Al to the doubling time of control cultures grown under the concentration in the soils (r 00.78, p<0.01). In contrast, the same conditions but in the absence of Al (Kunito et al. 2001). number of colonies on a diluted TSB agar plate without Al 5 8 Isolates were incubated in the diluted TSB with 0 or 1 mM (range, 3.4×10 to 1.5×10 per gram of dry soil) showed a Al at 25ºC, and the absorbances of the cultures at 660 nm were weaker correlation with exchangeable Al concentration in read at specific intervals. It should be mentioned that in this the soil (r 00.57, p<0.05). Due to obstruction by fungal evaluation, 1.5-fold-diluted TSB with 1 mM Al, instead of 15 growth, only 25 isolates were able to be single colony fold diluted TSB with 1.5 mM Al used for isolation, was purified from 8 soils (Table 2). In spite of the addition of employed. This is because 15-fold-diluted TSB was used to cycloheximide, only 11 isolates were bacteria, and 14 iso- isolate both oligotrophic and non-oligotrophic microorgan- lates were found to be yeasts. Among the 11 bacterial iso- isms, but all resultant isolates appeared not to be oligotroph, lates, the 16S rDNA sequences of 10 isolates showed high and because measuring a doubling time is easily conducted at similarity with species in Burkholderia. The same sequences 1.5-fold-diluted TSB with 1 mM Al due to a more rapid were found in strains BB4c and BB4d, BB5a and BB5c, and growth of the isolates in this condition. BB5d and BB5e (Fig. 1). The sequence similarities to Burkholderia phenazinium LMG 2247 were 99.1% for strain BA3a, 99.0% for BB4b, 99.1% for BB4c and BB4d, Results 99.1% for BB4e, 99.2% for BB5a and BB5c, and 99.4% for BB5d and BB5e. Strain BB4a showed 98.5, 98.4, 98.0, and Properties of soils are shown in Table 1. Exchangeable Al 97.8% sequence similarity to B. phenazinium LMG 2247 , −1 T T showed a wide range of concentrations (0.45–436 mg kg ). B. terricola LMG 20594 , B. sediminicola HU2-65W , and The Al level significantly increased with decreasing soil pH B. sartisoli RP007 , respectively. Our phylogenetic tree 7 x 2 (y03.27×10 ×0.0699 , r 00.94, p<0.001). shows that strain BB4a was closely related to B. sartisoli, Fig. 1 Neighbor-joining 52 BB5d, BB5e phylogenetic tree constructed BB5a, BB5c by using 16S rRNA gene B.phenazinium LMG2247 (U96936) 0.005 57 sequences. Bootstrap values BB4e (expressed as percentages of BB4c, BB4d DM-Ni1 (DQ419958) 1,000 replicates) greater than BA3a 50% are shown at branch points. DM-Cd1 (DQ419952) Pandoraea norimbergensis 61 DM-Co1(DQ419955) LMG 13019 (AF139171) was DM-Cd2 (DQ419953) used as an outgroup. Bar 0.005 BB4b substitutions per nucleotide BB4a position B.sartisoli RP007 (AF061872) B. sediminicola HU2-65W (EU035613) B.ginsengisoli KMY03 (AB201286) B.terricola LMG 20594 (AY040362) B.graminis C4D1M (U96939) B.fungorum LMG 16225 (AF215705) 75 T B. phytofirmans PsJN (AY497470) B.phymatum STM815 (AJ302312) 100 T B. caribiensis MWAP64 (Y17009) B.rhizoxinica HKI 454 (AJ938142) B. caryophylli ATCC 25418 (AB021423) B.cepacia ATCC 25416 (U96927) 98 T B. pseudomallei ATCC 23343 (DQ108392) B.glathei ATCC 29195 (AB021374) B. tropicalis Ppe8 (AJ420332) B.sacchari LMG19450 (AF263278) B. silvatlantica SRMrh-20 (AY965240) 66 T B.kururiensis KP23 (AB024310) B.acidipaludis SA33 (AB513180) P. norimbergensis LMG 13019 (AF139171) Ann Microbiol (2012) 62:1339–1344 1343 while the other strains were most closely related to B. The low diversity of Al-resistant bacteria, despite the phenazinium (Fig. 1). Strain BA1a was assigned to the long evolutionary time period of Al stress in these acid genus Acinetobacter. This strain was found to be most forest soils, might be due to the absence of specific genes closely related to A. guillouiae DSM 590 and A. berezinae for Al resistance in bacteria (Silver and Phung 2005). ATCC 17924 , with 16S rDNA sequence similarities of 99.4 Bacteria may have difficulty developing a specific Al resis- and 98.9%, respectively. It clustered with these two species tance system, because Al is present as insoluble forms at in our phylogenetic analysis (data not shown). around pH 7 in cytoplasm, as pointed out by Fischer et al. On the basis of ITS1, 5.8S rDNA, and ITS2 sequences, (2002). Hence, unspecific and passive Al resistance might 13 out of 14 yeast isolates were assigned to Lipomyces sp. be inherited as a species-specific characteristic; resistant (Table 2). The 5.8S rDNA sequence was identical among all species, usually present in low frequencies in soils without the Lipomyces isolates, and was also identical to the sequen- Al toxicity, might become dominant under Al stress. In T T ces of L. tetrasporus CBS 5910 and L. starkeyi CBS 1807 . contrast, diversity of bacteria resistant to heavy metals is The sequence of the ITS1 region in strains YA2a, YA2c, known to be very low in non-contaminated soils (e.g., YA2d, YA2e, YB3a, and YB3b had group I introns; the Kunito et al. 1997a), but many soil bacteria adapt to heavy conserved short sequences P, Q, R, and S (Cech 1988) were metal contamination by acquiring resistance through hori- also found in this region (data not shown). Strain YA2b zontal gene transfer, and diversity thus increases with time harbored an identical ITS1, 5.8S rDNA, and ITS2 sequence in contaminated soils (Kunito et al. 1998). to that of Cryptococcus podzolicus strain CBS 6819 , except Another Burkholderia sp., B. acidipaludis, has also been that YA2b had one insertion in the ITS1 region. isolated as an Al-resistant bacterium from acidic swamps In general, growth rates in diluted TSB were not signif- (Aizawa et al. 2010). Several heavy metal-resistant bacteria icantly changed by addition of Al for the Al-resistant yeasts, within Burkholderia (e.g., DM-Cd1 and DM-Ni1) were very but a longer doubling time was observed for the Al-resistant closely related to the Al-resistant bacteria that we isolated bacteria in diluted TSB with Al than for those without Al (Fig. 1). No published information was available for these (Table 2). The ratio of doubling time in diluted TSB with heavy metal-resistant bacteria; only their 16S rDNA sequen- 1 mM Al to that in diluted TSB without Al was significantly ces were deposited in GenBank/EMBL/DDBJ databases. larger for the bacteria (mean±SD, 1.31±0.10) than for the Meanwhile, several bacteria belonging to genera other than yeasts (1.05±0.02) (Welch’s t test, p<0.05), indicating a Burkholderia have been reported as Al-resistant: higher Al resistance in yeasts than bacteria. Although Al Arthrobacter sp. (Illmer and Mutschlechner 2004), levels were low in soil samples A1 and B1 (Table 1), the Flavobacterium sp. (Konishi et al. 1994), Pseudomonas resistance levels of the isolates were comparable with those fluorescens (Lemire et al. 2010), and some within the of strains isolated from soils with higher Al levels (Table 2). genera Acidocella, Acidiphilum, and Acidobacterium (Wakao et al. 2002). It thus appears that the species of Al- resistant bacterium occurring in a particular place is depen- Discussion dent on the soil types and land use patterns that usually determine bacterial community composition in soils. In contrast with our expectation, diversity of Al-resistant It should be mentioned that isolation of the Al-resistant bacteria was not related to Al concentrations in soils, and Lipomyces sp., albeit with cycloheximide added to the me- almost all bacteria found were assigned to the genus dium, was owing to its resistance to both cycloheximide and Burkholderia (Table 2). In addition, the Al resistance levels acidity (Naganuma et al. 1999; Müller et al. 2007). In our of isolates, evaluated using a change in doubling time by Al previous studies, Lipomyces sp. were not isolated from soils addition, were not associated with Al concentrations in using a medium with added cycloheximide and Cu or Zn soils. (Kunito et al. 1997a, b, 2001). Because the pH of the According to Saigusa et al. (1980), Al-sensitive plants begin medium was not acidic in our previous studies, it seems to become injured when the concentration of exchangeable likely that the medium may need to be acidified for the −1 Al exceeds 90 mg kg dry soil. Five out of 8 of our soils isolation of Lipomyces sp. Alternatively, Lipomyces sp. exhibited higher Al levels than this threshold level (Table 1). may exhibit Al resistance, but be Cu and Zn sensitive. It is also known that microorganisms are more sensitive to Similarly, a Cryptococcus sp. was isolated from acidic tea Al toxicity than are trees (Joner et al. 2005). However, field soil as an Al-resistant yeast, using an acidic medium populations of Al-resistant microorganisms increased with with added cycloheximide (Kanazawa et al. 2005). Hence, Al levels in the soils investigated. Al-resistant microorganisms some species and/or strains of Cryptococcus may have are thus of potential importance as contributors to biological resistance to both cycloheximide and acidity, and also to processes in acid soils with high Al levels. Al, in a similar manner to Lipomyces sp. 1344 Ann Microbiol (2012) 62:1339–1344 Kunito T, Nagaoka K, Tada N, Saeki K, Senoo K, Oyaizu H, Matsumoto Our study has revealed that populations of Al-resistant S (1997b) Characterization of Cu-resistant bacterial communities in microorganisms increased with increasing Al levels in acid- Cu-contaminated soils. Soil Sci Plant Nutr 43:709–717 ic forest soils, although Al levels were greater than the Kunito T, Oyaizu H, Matsumoto S (1998) Ecology of soil heavy metal- threshold of toxicity to plants. Hence, such Al-resistant resistant bacteria and perspective of bioremediation of heavy metal- contaminated soils. Recent Res Devel Agric Biol Chem 2:185–206 microorganisms may play a significant role in the biological Kunito T, Saeki K, Nagaoka K, Oyaizu H, Matsumoto S (2001) Charac- processes within acid soils with high Al levels. The low terization of copper-resistant bacterial community in rhizosphere of diversity of resistant microorganisms might, however, cause highly copper-contaminated soil. Eur J Soil Biol 37:95–102 their low functional diversity. Further studies are needed to Kunito T, Akagi Y, Park H-D, Toda H (2009) Influences of nitrogen and phosphorus addition on polyphenol oxidase activity in a evaluate their possible roles in nutrient cycling and ecosystem forested Andisol. Eur J For Res 128:361–366 function in acidic forest soils with high Al levels. Kunito T, Tsunekawa M, Yoshida S, Park H-D, Toda H, Nagaoka K, Saeki K (2011) Soil properties affecting phosphorus forms and Acknowledgements We thank I. Isomura for technical assistance. phosphatase activities in Japanese forest soils: Soil microorgan- This study was supported by Grants-in-Aid for Young Scientists (B) isms may be limited by phosphorus. Soil Sci, doi:10.1097/ (No. 16710002 and 19710008 to T. Kunito) from the Ministry of SS.0b013e3182378153 Education, Culture, Sports, Science and Technology, Japan. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948 References Lemire J, Auger C, Bignucolo A, Appanna VP, Appanna VD (2010) Metabolic strategies deployed by Pseudomonas fluorescens to Aizawa T, Ve NB, Vijarnsorn P, Nakajima M, Sunairi M (2010) combat metal pollutants: biotechnological prospects. In: Mén- Burkholderia acidipaludis sp. nov., aluminium-tolerant bacteria dez-Vilas A (ed) Current Research. Technology and Education isolated from Chinese water chestnut (Eleocharis dulcis) growing Topics in Applied Microbiology and Microbial Biotechnology, in highly acidic swamps in South-East Asia. Int J Syst Evol Formatex Research Centre, Badajoz, Spain, pp 177–187 Microbiol 60:2036–2041 Macdonald TL, Martin RB (1988) Aluminum ion in biological systems. Cech TR (1988) Conserved sequences and structures of group I introns: Trends Biochem Sci 13:15–19 building an active site for RNA catalysis: a review. Gene 73:259–271 Makimura K, Murayama YS, Yamaguchi H (1994) Detection of a wide Exley C, Birchall JD (1992) The cellular toxicity of aluminium. J range of medically important fungal species by polymerase chain Theor Biol 159:83–98 reaction (PCR). J Med Microbiol 40:358–364 Fischer J, Quentmeier A, Gansel S, Sabados V, Friedrich CG (2002) Inducible Matsumoto H (2000) Cell biology of aluminum toxicity and tolerance aluminum resistance of Acidiphilium cryptum and aluminum toler- in higher plants. Int Rev Cytol 200:1–46 ance of other acidophilic bacteria. Arch Microbiol 178:554–558 Müller WJ, Albertyn J, Smit MS (2007) Cycloheximide resistance in Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for the Lipomycetaceae revisited. Can J Microbiol 53:509–513 basidiomycetes: application to the identification of mycorrhizae Naganuma T, Katsumata K, Ando T, Watanabe H, Nishimura K, Uzuka and rusts. Mol Ecol 2:113–118 Y (1999) An improved method for isolating yeasts in the genus Godbold DL, Fritz E, Hüttermann A (1988) Aluminum toxicity and Lipomyces and related genera from soil. Biosci Biotech Biochem forest decline. Proc Natl Acad Sci USA 85:3888–3892 63:195–198 Hamel R, Appanna VD (2003) Aluminum detoxification in Pseudo- Piña RG, Cervantes C (1996) Microbial interactions with aluminium. monas fluorescens is mediated by oxalate and phosphatidyletha- BioMetals 9:311–316 nolamine. Biochim Biophys Acta 1619:70–76 Robert M (1995) Aluminum toxicity: a major stress for microbes in the Illmer P, Mutschlechner W (2004) Effect of temperature and pH on the environment. In: Huang PM, Berthelin J, Bollag J-M, McGill toxicity of aluminium towards two new, soil born species of WB, Page AL (eds) Environmental impact of soil component Arthrobacter sp. J Basic Microbiol 2:98–105 interactions: metals, other inorganics, and microbial activities. Illmer P, Marschall K, Schinner F (1995) Influence of available aluminium CRC Press, Boca Raton, pp 227–242 on soil micro-organisms. Lett Appl Microbiol 21:393–397 Saigusa M, Shoji S, Takahashi T (1980) Plant root growth in acid Andosols Illmer P, Obertegger U, Schinner F (2003) Microbiological properties from northeastern Japan: 2. Exchange acidity Y as a realistic measure in acidic forest soils with special consideration of KCl extractable of aluminum toxicity potential. Soil Sci 130:242–250 Al. Wat Air Soil Pollut 148:3–14 Silver S, Phung LT (2005) A bacterial view of the periodic table: genes Joner EJ, Eldhuset TD, Lange H, Frostegård Å (2005) Changes in the and proteins for toxic inorganic ions. J Ind Microbiol Biotechnol microbial community in a forest soil amended with aluminium in 32:587–605 situ. Plant Soil 275:295–304 Sposito G (1996) The Environmental Chemistry of Aluminum, 2nd Kanazawa S, Kunito T (1996) Preparation of pH 3.0 agar plate, edn. CRC Press, Boca Raton enumeration of acid-tolerant, and Al-resistant microorganisms in Tago K, Sekiya E, Kiho A, Katsuyama C, Hoshito Y, Yamada N, acid soils. Soil Sci Plant Nutr 42:165–173 Hirano K, Sawada H, Hayatsu M (2006) Diversity of Kanazawa S, Chau NTT, Miyaki S (2005) Identification and charac- fenitrothion-degrading bacteria in soils from distant geographic terization of high acid tolerant and aluminum resistant yeasts areas. Microbes Environ 21:58–64 isolated from tea soils. Soil Sci Plant Nutr 51:671–674 Wakao N, Yasuda T, Jojima Y, Yamanaka S, Hiraishi A (2002) Enhanced Konishi S, Souta I, Takahashi J, Ohmoto M, Kaneko S (1994) Isolation growth of Acidocella facilis and related acidophilic bacteria at and characteristics of acid- and aluminum-tolerant bacterium. high concentrations of aluminum. Microbes Environ 17:98–104 Biosci Biotech Biochem 58:1960–1963 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct Kunito T, Shibata S, Matsumoto S, Oyaizu H (1997a) Zinc resistance sequencing of fungal ribosomal RNA genes for phylogenetics. In: of Methylobacterium species. Biosci Biotech Biochem 61:729– Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a 731 guide to methods and applications. Academic, London, pp 315–322 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Genera Burkholderia and Lipomyces are predominant aluminum-resistant microorganisms isolated from acidic forest soils using cycloheximide-amended growth media

Download PDF
6 pages

Loading next page...
 
/lp/springer-journals/genera-burkholderia-and-lipomyces-are-predominant-aluminum-resistant-AAKdtjYqMj

References (43)

Publisher
Springer Journals
Copyright
Copyright © 2011 by Springer-Verlag and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-011-0393-4
Publisher site
See Article on Publisher Site

Abstract

Ann Microbiol (2012) 62:1339–1344 DOI 10.1007/s13213-011-0393-4 SHORT COMMUNICATION Genera Burkholderia and Lipomyces are predominant aluminum-resistant microorganisms isolated from acidic forest soils using cycloheximide-amended growth media Takashi Kunito & Miki Owaki & Yasutaka Ihyo & Hirotaka Sumi & Hideshige Toda & Daisuke Fukuda & Ho-Dong Park Received: 20 September 2011 /Accepted: 15 November 2011 /Published online: 29 November 2011 # Springer-Verlag and the University of Milan 2011 . . . Abstract In acidic forest soils, microorganisms should Keywords Aluminum Burkholderia Forest soil adapt to toxicity of aluminum (Al), which is solubilized by Lipomyces Resistance acidity. We hypothesized that Al-resistant bacteria are diverse, especially in soils with high levels of Al, because these bacteria are expected to have adapted to Al stress over a Introduction long time. We isolated Al-resistant bacteria from acid forest soils using diluted tryptic soy broth agar plates with added Al Aluminum has a higher affinity for phosphorus (P) and and cycloheximide, and examined the relationship between slower reaction kinetics than calcium (Ca) or magnesium their diversity and Al levels in soils. Based on 16S rDNA (Mg), and is thus a powerful inhibitor of many biological sequences, 10 out of 11 isolated bacteria were assigned to the processes dependent on Ca and Mg (Macdonald and Martin genus Burkholderia, and 1 to the genus Acinetobacter. 1988; Exley and Birchall 1992). Aluminum toxicity is a Although cycloheximide was added to the Al-enriched agar, major problem in acid soils because Al solubility increases yeasts were isolated from soils, and were examined. On the with decreasing soil pH (Sposito 1996). Acidic precipitation basis of ITS1, 5.8S rDNA, and ITS2 sequences, 13 out of 14 has therefore exaggerated the adverse effects of Al on plants yeast isolates were assigned to Lipomyces sp. and 1 isolate and microorganisms in these soils. High levels of Al in soils to Cryptococcus sp. Diversity of Al-resistant bacteria was may cause root damage and mineral imbalances in trees, and low in acidic forest soils, and was not related to Al levels in even result in forest decline (Godbold et al. 1988). soils. Population numbers of Al-resistant microorganisms, Microorganisms are known to be more sensitive to Al tox- however, increased with increasing Al levels. icity than are trees (Joner et al. 2005). However, few studies have assessed the adverse effects of Al on soil microorgan- isms or their adaptation to Al stress (Piña and Cervantes 1996). In contrast, the environmental behavior of Al in soils : : : : : T. Kunito (*) M. Owaki Y. Ihyo H. Sumi H. Toda (Sposito 1996), and the adaptation of plants to Al toxicity H.-D. Park Department of Environmental Sciences, Faculty of Science, (Matsumoto 2000), have been well studied. Aluminum Shinshu University, directly affects microorganisms through its fixation to cell 3-1-1 Asahi, walls, fixation to DNA via binding with P, and its toxic Matsumoto 390-8621, Japan effects on enzymes (Robert 1995). It indirectly affects e-mail: kunito@shinshu-u.ac.jp microorganisms through perturbation of Ca and Mg metab- D. Fukuda olism (Robert 1995), leading to reductions in microbial Daiichi Sankyo Co. Ltd, biomass, basal respiration, ATP levels, and protease activity 1-2-58 Hiromachi, in soils (Illmer et al. 2003). Al-resistant microorganisms are Shinagawa-ku, Tokyo 140-8710, Japan 1340 Ann Microbiol (2012) 62:1339–1344 therefore likely to play a key role in various microbially- soils were classified as Haplic Brown Forest soils mediated processes such as nutrient cycling in soils. (Inceptisols). Vegetation was predominately larch, Larix Bacterial Al resistance systems seem to be unspecific and kaempferi, in five sites (A1, A3, B1, B2, and B3), passive (meaning that systems have another primary role, Japanese red pine, Pinus densiflora, in one site (A2), such as production of extracellular polysaccharides; Robert Japanese chestnut, Castanea crenata, and Mongolian oak, 1995). Bacteria have, however, developed sophisticated re- Quercus mongolica, in one site (B4), and Veitch fir, Abies sistance mechanisms to various heavy metals, including veitchii, and Maries fir, Abies mariesii, in one site (B5). efflux systems for cadmium (Cd), lead (Pb), copper (Cu), Sampling sites were located at altitudes between 480 and and zinc (Zn), and volatilization of mercury (Hg) (Silver and 2,050 m. The mean annual precipitation at meteorological Phung 2005). No specific genes for Al resistance have so far stations adjacent to sampling sites ranged from 941 to been found in bacteria (Silver and Phung 2005). The most 2,152 mm, and the air temperature from 7.1 to 16.5°C. extensively investigated is Pseudomonas fluorescens,in Each soil sample was sieved through a 2-mm mesh and which Al is secreted in association with oxalate and phos- homogenized well. A portion of each sample was air-dried for phatidylethanolamine by an energy-independent process chemical analysis, while the remainder was kept field-moist at (neither proton-motive force nor ATP hydrolysis), and is 4°C. Soil properties were determined using standard methods immobilized in the insoluble gelatinous precipitate (Hamel as described elsewhere (Kunito et al. 2009, 2011); these are and Appanna 2003; Lemire et al. 2010). shown in Table 1. Exchangeable Al, a fraction toxic to Acidic forest soils with high exchangeable Al concentra- microbes (Illmer et al. 1995) and plants (Saigusa et al. tions are widely distributed in Japan, and have developed 1980), was extracted using 1 M KCl, and analyzed using through natural processes across long periods of time. We an atomic absorption spectrometer 5100ZL (Perkin Elmer, hypothesized that Al-resistant bacteria are diverse, especially Tokyo). All data are expressed on a dry weight basis. in soils with high levels of Al, because these bacteria are expected to have adapted to Al stress in acidic forest soils over Isolation and identification of Al-resistant microorganisms a long time. To examine this, we isolated and identified pre- dominant Al-resistant bacteria from Japanese acidic forest soils. Moist soil samples were dispersed in sterile tap water using a Waring blender 500 C (Sakuma, Tokyo), and the resulting slurry was decimally diluted with sterile tap water. Samples Materials and methods were spread on a 15-fold-diluted TSB agar plate (tryptic soy broth, 2 g; agar, 10 g; cycloheximide, 50 mg; distilled water, Soils 1 L; pH 4.0), with added Al at 1.5 mM. The Al level in the medium selected about 10% of the microbial population Soil samples were collected from A horizons in forests in growing on a 15-fold-diluted TSB agar plate (pH 4.0) with- Nagano Prefecture, Japan. Three soils were classified as out Al. For preparation of the diluted TSB agar plate, an allophanic Kuroboku soils (allophanic Andisols), and five acidified TSB medium with HCl, an AlCl solution, and an Table 1 Properties of forest soils used Soil no. Latitude Longitude Altitude Soil order Soil group (Japan) pH Organic Total N Exchangeable −1 (N) (E) (m) (USDA) C (%) (%) Al (mg kg ) A1 36°24′ 138°03′ 1,510 Andisol Allophanic 6.1 5.2 0.35 1.06 Kuroboku soil A2 35°55′ 138°02′ 880 Andisol Allophanic 5.2 8.2 0.69 56.7 Kuroboku soil A3 35°50′ 137°52′ 1,300 Andisol Allophanic 4.9 8.6 0.54 145 Kuroboku soil B1 36°31′ 138°10′ 480 Inceptisol Haplic Brown 6.9 2.9 0.23 0.45 Forest soil B2 35°51′ 137°50′ 1,560 Inceptisol Haplic Brown 4.9 8.8 0.69 104 Forest soil B3 35°50′ 137°50′ 1,870 Inceptisol Haplic Brown 4.4 10.2 0.69 146 Forest soil B4 36°26′ 138°20′ 1,100 Inceptisol Haplic Brown 4.5 18.0 1.0 217 Forest soil B5 35°50′ 137°49′ 2,050 Inceptisol Haplic Brown 4.1 21.9 1.1 436 Forest soil Ann Microbiol (2012) 62:1339–1344 1341 agar solution were separately autoclaved to prevent hydro- solution, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% lysis of the agar (Kanazawa and Kunito 1996). After cooling Triton X-100, 1.5 mM MgCl , 0.2 mM of each dNTP, and to 50ºC, these three solutions were mixed, and filter- 1 U of ExTaq polymerase (Takara Bioscience, Tokyo). sterilized cycloheximide was added. The pH of the resulting Amplification was carried out over 30 cycles (denaturation agar plate was confirmed as 4.0 in a preliminary experiment. at 95ºC for 30 s, annealing at 55ºC for 30 s, and extension at After 10 days incubation at 25ºC, five colonies were ran- 72ºC for 1 min). Amplified fragments of 16S rDNA were domly isolated from each soil sample, and subjected to purified using ExoSAP-IT (GE Healthcare Bioscience, single colony isolation. Single colony isolation, however, Tokyo), and were sequenced directly. Although cyclohexi- was only partially successful (Table 2) because of obstruc- mide, an inhibitor of cytosolic protein biosynthesis in eukar- tion by fungal growth on the plate, despite the addition of yotes, was added to the Al-enriched agar, yeasts and not cycloheximide. bacteria were isolated from four soils (Table 2). These iso- Genomic DNA of isolated bacteria was extracted lates were identified based on ITS1, 5.8S rDNA, and ITS2 according to Tago et al. (2006), and the isolates sequences. These were amplified using primers ITS1-F were identified based on 16S rRNA gene sequences. These (Gardes and Bruns 1993) and ITS4 (White et al. 1990) after were amplified by PCR using universal primers; 27f extraction of nuclear DNA by the method of Makimura et al. (5′-AGAGTTTGATCATGGCTCAG-3′) and 1492r (5′- (1994). The PCR protocol and sequence analysis were as GGCTACCTTGTTACGACTT-3′). PCR was performed in described above. Phylogenetic analysis was performed using 10 μl of a mixture containing 1 μl of genomic DNA CLUSTAL X version 2 (Larkin et al. 2007). Table 2 Characteristics of aluminum-resistant bacteria and yeasts from forest soils Isolates Soil no. Accession no. Tentative identification Doubling time (hr) Ratio of doubling time (Diluted TSB+1 mM Al) / (Diluted TSB) Diluted TSB Diluted TSB+1 mM Al Bacteria BA1a A1 AB665296 Acinetobacter sp. 7.20 7.47 1.04 BA3a A3 AB665286 Burkholderia sp. 3.31 4.19 1.27 BB4a B4 AB665287 Burkholderia sp. 2.77 3.48 1.26 BB4b B4 AB665288 Burkholderia sp. 2.86 3.12 1.09 BB4c B4 AB665289 Burkholderia sp. 3.15 4.89 1.55 BB4d B4 AB665290 Burkholderia sp. 3.30 5.56 1.68 BB4e B4 AB665291 Burkholderia sp. 3.14 6.33 2.02 BB5a B5 AB665292 Burkholderia sp. 3.35 3.51 1.05 BB5c B5 AB665293 Burkholderia sp. 3.04 3.31 1.09 BB5d B5 AB665294 Burkholderia sp. 2.73 3.35 1.23 BB5e B5 AB665295 Burkholderia sp. 2.69 3.08 1.14 Yeasts YA2a A2 AB665297 Lipomyces sp. 7.99 7.62 0.95 YA2b A2 AB665310 Cryptococcus sp. 4.67 4.23 0.91 YA2c A2 AB665298 Lipomyces sp. 6.42 7.14 1.11 YA2d A2 AB665299 Lipomyces sp. 6.01 6.46 1.07 YA2e A2 AB665300 Lipomyces sp. 5.33 5.45 1.02 YB1a B1 AB665301 Lipomyces sp. 6.83 6.51 0.95 YB1b B1 AB665302 Lipomyces sp. 6.92 7.49 1.08 YB2a B2 AB665303 Lipomyces sp. 6.45 6.32 0.98 YB2b B2 AB665304 Lipomyces sp. 6.03 6.19 1.03 YB2c B2 AB665305 Lipomyces sp. 6.42 6.56 1.02 YB2d B2 AB665306 Lipomyces sp. 6.20 5.83 0.94 YB2e B2 AB665307 Lipomyces sp. 6.26 6.22 0.99 YB3a B3 AB665308 Lipomyces sp. 6.17 6.99 1.13 YB3b B3 AB665309 Lipomyces sp. 5.02 7.17 1.43 TSB, tryptic soy broth 1342 Ann Microbiol (2012) 62:1339–1344 Evaluation of Al resistance of isolates The numbers of colonies on diluted TSB agar plates with 4 6 1.5 mM Al added ranged from 3.9×10 to 4.6×10 per gram Levels of Al resistance were evaluated using a ratio of of dry soil, with a significant positive correlation between doubling time in 1.5-fold-diluted TSB (pH 4.0) amended with the number of Al-resistant colonies and the exchangeable Al Al to the doubling time of control cultures grown under the concentration in the soils (r 00.78, p<0.01). In contrast, the same conditions but in the absence of Al (Kunito et al. 2001). number of colonies on a diluted TSB agar plate without Al 5 8 Isolates were incubated in the diluted TSB with 0 or 1 mM (range, 3.4×10 to 1.5×10 per gram of dry soil) showed a Al at 25ºC, and the absorbances of the cultures at 660 nm were weaker correlation with exchangeable Al concentration in read at specific intervals. It should be mentioned that in this the soil (r 00.57, p<0.05). Due to obstruction by fungal evaluation, 1.5-fold-diluted TSB with 1 mM Al, instead of 15 growth, only 25 isolates were able to be single colony fold diluted TSB with 1.5 mM Al used for isolation, was purified from 8 soils (Table 2). In spite of the addition of employed. This is because 15-fold-diluted TSB was used to cycloheximide, only 11 isolates were bacteria, and 14 iso- isolate both oligotrophic and non-oligotrophic microorgan- lates were found to be yeasts. Among the 11 bacterial iso- isms, but all resultant isolates appeared not to be oligotroph, lates, the 16S rDNA sequences of 10 isolates showed high and because measuring a doubling time is easily conducted at similarity with species in Burkholderia. The same sequences 1.5-fold-diluted TSB with 1 mM Al due to a more rapid were found in strains BB4c and BB4d, BB5a and BB5c, and growth of the isolates in this condition. BB5d and BB5e (Fig. 1). The sequence similarities to Burkholderia phenazinium LMG 2247 were 99.1% for strain BA3a, 99.0% for BB4b, 99.1% for BB4c and BB4d, Results 99.1% for BB4e, 99.2% for BB5a and BB5c, and 99.4% for BB5d and BB5e. Strain BB4a showed 98.5, 98.4, 98.0, and Properties of soils are shown in Table 1. Exchangeable Al 97.8% sequence similarity to B. phenazinium LMG 2247 , −1 T T showed a wide range of concentrations (0.45–436 mg kg ). B. terricola LMG 20594 , B. sediminicola HU2-65W , and The Al level significantly increased with decreasing soil pH B. sartisoli RP007 , respectively. Our phylogenetic tree 7 x 2 (y03.27×10 ×0.0699 , r 00.94, p<0.001). shows that strain BB4a was closely related to B. sartisoli, Fig. 1 Neighbor-joining 52 BB5d, BB5e phylogenetic tree constructed BB5a, BB5c by using 16S rRNA gene B.phenazinium LMG2247 (U96936) 0.005 57 sequences. Bootstrap values BB4e (expressed as percentages of BB4c, BB4d DM-Ni1 (DQ419958) 1,000 replicates) greater than BA3a 50% are shown at branch points. DM-Cd1 (DQ419952) Pandoraea norimbergensis 61 DM-Co1(DQ419955) LMG 13019 (AF139171) was DM-Cd2 (DQ419953) used as an outgroup. Bar 0.005 BB4b substitutions per nucleotide BB4a position B.sartisoli RP007 (AF061872) B. sediminicola HU2-65W (EU035613) B.ginsengisoli KMY03 (AB201286) B.terricola LMG 20594 (AY040362) B.graminis C4D1M (U96939) B.fungorum LMG 16225 (AF215705) 75 T B. phytofirmans PsJN (AY497470) B.phymatum STM815 (AJ302312) 100 T B. caribiensis MWAP64 (Y17009) B.rhizoxinica HKI 454 (AJ938142) B. caryophylli ATCC 25418 (AB021423) B.cepacia ATCC 25416 (U96927) 98 T B. pseudomallei ATCC 23343 (DQ108392) B.glathei ATCC 29195 (AB021374) B. tropicalis Ppe8 (AJ420332) B.sacchari LMG19450 (AF263278) B. silvatlantica SRMrh-20 (AY965240) 66 T B.kururiensis KP23 (AB024310) B.acidipaludis SA33 (AB513180) P. norimbergensis LMG 13019 (AF139171) Ann Microbiol (2012) 62:1339–1344 1343 while the other strains were most closely related to B. The low diversity of Al-resistant bacteria, despite the phenazinium (Fig. 1). Strain BA1a was assigned to the long evolutionary time period of Al stress in these acid genus Acinetobacter. This strain was found to be most forest soils, might be due to the absence of specific genes closely related to A. guillouiae DSM 590 and A. berezinae for Al resistance in bacteria (Silver and Phung 2005). ATCC 17924 , with 16S rDNA sequence similarities of 99.4 Bacteria may have difficulty developing a specific Al resis- and 98.9%, respectively. It clustered with these two species tance system, because Al is present as insoluble forms at in our phylogenetic analysis (data not shown). around pH 7 in cytoplasm, as pointed out by Fischer et al. On the basis of ITS1, 5.8S rDNA, and ITS2 sequences, (2002). Hence, unspecific and passive Al resistance might 13 out of 14 yeast isolates were assigned to Lipomyces sp. be inherited as a species-specific characteristic; resistant (Table 2). The 5.8S rDNA sequence was identical among all species, usually present in low frequencies in soils without the Lipomyces isolates, and was also identical to the sequen- Al toxicity, might become dominant under Al stress. In T T ces of L. tetrasporus CBS 5910 and L. starkeyi CBS 1807 . contrast, diversity of bacteria resistant to heavy metals is The sequence of the ITS1 region in strains YA2a, YA2c, known to be very low in non-contaminated soils (e.g., YA2d, YA2e, YB3a, and YB3b had group I introns; the Kunito et al. 1997a), but many soil bacteria adapt to heavy conserved short sequences P, Q, R, and S (Cech 1988) were metal contamination by acquiring resistance through hori- also found in this region (data not shown). Strain YA2b zontal gene transfer, and diversity thus increases with time harbored an identical ITS1, 5.8S rDNA, and ITS2 sequence in contaminated soils (Kunito et al. 1998). to that of Cryptococcus podzolicus strain CBS 6819 , except Another Burkholderia sp., B. acidipaludis, has also been that YA2b had one insertion in the ITS1 region. isolated as an Al-resistant bacterium from acidic swamps In general, growth rates in diluted TSB were not signif- (Aizawa et al. 2010). Several heavy metal-resistant bacteria icantly changed by addition of Al for the Al-resistant yeasts, within Burkholderia (e.g., DM-Cd1 and DM-Ni1) were very but a longer doubling time was observed for the Al-resistant closely related to the Al-resistant bacteria that we isolated bacteria in diluted TSB with Al than for those without Al (Fig. 1). No published information was available for these (Table 2). The ratio of doubling time in diluted TSB with heavy metal-resistant bacteria; only their 16S rDNA sequen- 1 mM Al to that in diluted TSB without Al was significantly ces were deposited in GenBank/EMBL/DDBJ databases. larger for the bacteria (mean±SD, 1.31±0.10) than for the Meanwhile, several bacteria belonging to genera other than yeasts (1.05±0.02) (Welch’s t test, p<0.05), indicating a Burkholderia have been reported as Al-resistant: higher Al resistance in yeasts than bacteria. Although Al Arthrobacter sp. (Illmer and Mutschlechner 2004), levels were low in soil samples A1 and B1 (Table 1), the Flavobacterium sp. (Konishi et al. 1994), Pseudomonas resistance levels of the isolates were comparable with those fluorescens (Lemire et al. 2010), and some within the of strains isolated from soils with higher Al levels (Table 2). genera Acidocella, Acidiphilum, and Acidobacterium (Wakao et al. 2002). It thus appears that the species of Al- resistant bacterium occurring in a particular place is depen- Discussion dent on the soil types and land use patterns that usually determine bacterial community composition in soils. In contrast with our expectation, diversity of Al-resistant It should be mentioned that isolation of the Al-resistant bacteria was not related to Al concentrations in soils, and Lipomyces sp., albeit with cycloheximide added to the me- almost all bacteria found were assigned to the genus dium, was owing to its resistance to both cycloheximide and Burkholderia (Table 2). In addition, the Al resistance levels acidity (Naganuma et al. 1999; Müller et al. 2007). In our of isolates, evaluated using a change in doubling time by Al previous studies, Lipomyces sp. were not isolated from soils addition, were not associated with Al concentrations in using a medium with added cycloheximide and Cu or Zn soils. (Kunito et al. 1997a, b, 2001). Because the pH of the According to Saigusa et al. (1980), Al-sensitive plants begin medium was not acidic in our previous studies, it seems to become injured when the concentration of exchangeable likely that the medium may need to be acidified for the −1 Al exceeds 90 mg kg dry soil. Five out of 8 of our soils isolation of Lipomyces sp. Alternatively, Lipomyces sp. exhibited higher Al levels than this threshold level (Table 1). may exhibit Al resistance, but be Cu and Zn sensitive. It is also known that microorganisms are more sensitive to Similarly, a Cryptococcus sp. was isolated from acidic tea Al toxicity than are trees (Joner et al. 2005). However, field soil as an Al-resistant yeast, using an acidic medium populations of Al-resistant microorganisms increased with with added cycloheximide (Kanazawa et al. 2005). Hence, Al levels in the soils investigated. Al-resistant microorganisms some species and/or strains of Cryptococcus may have are thus of potential importance as contributors to biological resistance to both cycloheximide and acidity, and also to processes in acid soils with high Al levels. Al, in a similar manner to Lipomyces sp. 1344 Ann Microbiol (2012) 62:1339–1344 Kunito T, Nagaoka K, Tada N, Saeki K, Senoo K, Oyaizu H, Matsumoto Our study has revealed that populations of Al-resistant S (1997b) Characterization of Cu-resistant bacterial communities in microorganisms increased with increasing Al levels in acid- Cu-contaminated soils. Soil Sci Plant Nutr 43:709–717 ic forest soils, although Al levels were greater than the Kunito T, Oyaizu H, Matsumoto S (1998) Ecology of soil heavy metal- threshold of toxicity to plants. Hence, such Al-resistant resistant bacteria and perspective of bioremediation of heavy metal- contaminated soils. Recent Res Devel Agric Biol Chem 2:185–206 microorganisms may play a significant role in the biological Kunito T, Saeki K, Nagaoka K, Oyaizu H, Matsumoto S (2001) Charac- processes within acid soils with high Al levels. The low terization of copper-resistant bacterial community in rhizosphere of diversity of resistant microorganisms might, however, cause highly copper-contaminated soil. Eur J Soil Biol 37:95–102 their low functional diversity. Further studies are needed to Kunito T, Akagi Y, Park H-D, Toda H (2009) Influences of nitrogen and phosphorus addition on polyphenol oxidase activity in a evaluate their possible roles in nutrient cycling and ecosystem forested Andisol. Eur J For Res 128:361–366 function in acidic forest soils with high Al levels. Kunito T, Tsunekawa M, Yoshida S, Park H-D, Toda H, Nagaoka K, Saeki K (2011) Soil properties affecting phosphorus forms and Acknowledgements We thank I. Isomura for technical assistance. phosphatase activities in Japanese forest soils: Soil microorgan- This study was supported by Grants-in-Aid for Young Scientists (B) isms may be limited by phosphorus. Soil Sci, doi:10.1097/ (No. 16710002 and 19710008 to T. Kunito) from the Ministry of SS.0b013e3182378153 Education, Culture, Sports, Science and Technology, Japan. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948 References Lemire J, Auger C, Bignucolo A, Appanna VP, Appanna VD (2010) Metabolic strategies deployed by Pseudomonas fluorescens to Aizawa T, Ve NB, Vijarnsorn P, Nakajima M, Sunairi M (2010) combat metal pollutants: biotechnological prospects. In: Mén- Burkholderia acidipaludis sp. nov., aluminium-tolerant bacteria dez-Vilas A (ed) Current Research. Technology and Education isolated from Chinese water chestnut (Eleocharis dulcis) growing Topics in Applied Microbiology and Microbial Biotechnology, in highly acidic swamps in South-East Asia. Int J Syst Evol Formatex Research Centre, Badajoz, Spain, pp 177–187 Microbiol 60:2036–2041 Macdonald TL, Martin RB (1988) Aluminum ion in biological systems. Cech TR (1988) Conserved sequences and structures of group I introns: Trends Biochem Sci 13:15–19 building an active site for RNA catalysis: a review. Gene 73:259–271 Makimura K, Murayama YS, Yamaguchi H (1994) Detection of a wide Exley C, Birchall JD (1992) The cellular toxicity of aluminium. J range of medically important fungal species by polymerase chain Theor Biol 159:83–98 reaction (PCR). J Med Microbiol 40:358–364 Fischer J, Quentmeier A, Gansel S, Sabados V, Friedrich CG (2002) Inducible Matsumoto H (2000) Cell biology of aluminum toxicity and tolerance aluminum resistance of Acidiphilium cryptum and aluminum toler- in higher plants. Int Rev Cytol 200:1–46 ance of other acidophilic bacteria. Arch Microbiol 178:554–558 Müller WJ, Albertyn J, Smit MS (2007) Cycloheximide resistance in Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for the Lipomycetaceae revisited. Can J Microbiol 53:509–513 basidiomycetes: application to the identification of mycorrhizae Naganuma T, Katsumata K, Ando T, Watanabe H, Nishimura K, Uzuka and rusts. Mol Ecol 2:113–118 Y (1999) An improved method for isolating yeasts in the genus Godbold DL, Fritz E, Hüttermann A (1988) Aluminum toxicity and Lipomyces and related genera from soil. Biosci Biotech Biochem forest decline. Proc Natl Acad Sci USA 85:3888–3892 63:195–198 Hamel R, Appanna VD (2003) Aluminum detoxification in Pseudo- Piña RG, Cervantes C (1996) Microbial interactions with aluminium. monas fluorescens is mediated by oxalate and phosphatidyletha- BioMetals 9:311–316 nolamine. Biochim Biophys Acta 1619:70–76 Robert M (1995) Aluminum toxicity: a major stress for microbes in the Illmer P, Mutschlechner W (2004) Effect of temperature and pH on the environment. In: Huang PM, Berthelin J, Bollag J-M, McGill toxicity of aluminium towards two new, soil born species of WB, Page AL (eds) Environmental impact of soil component Arthrobacter sp. J Basic Microbiol 2:98–105 interactions: metals, other inorganics, and microbial activities. Illmer P, Marschall K, Schinner F (1995) Influence of available aluminium CRC Press, Boca Raton, pp 227–242 on soil micro-organisms. Lett Appl Microbiol 21:393–397 Saigusa M, Shoji S, Takahashi T (1980) Plant root growth in acid Andosols Illmer P, Obertegger U, Schinner F (2003) Microbiological properties from northeastern Japan: 2. Exchange acidity Y as a realistic measure in acidic forest soils with special consideration of KCl extractable of aluminum toxicity potential. Soil Sci 130:242–250 Al. Wat Air Soil Pollut 148:3–14 Silver S, Phung LT (2005) A bacterial view of the periodic table: genes Joner EJ, Eldhuset TD, Lange H, Frostegård Å (2005) Changes in the and proteins for toxic inorganic ions. J Ind Microbiol Biotechnol microbial community in a forest soil amended with aluminium in 32:587–605 situ. Plant Soil 275:295–304 Sposito G (1996) The Environmental Chemistry of Aluminum, 2nd Kanazawa S, Kunito T (1996) Preparation of pH 3.0 agar plate, edn. CRC Press, Boca Raton enumeration of acid-tolerant, and Al-resistant microorganisms in Tago K, Sekiya E, Kiho A, Katsuyama C, Hoshito Y, Yamada N, acid soils. Soil Sci Plant Nutr 42:165–173 Hirano K, Sawada H, Hayatsu M (2006) Diversity of Kanazawa S, Chau NTT, Miyaki S (2005) Identification and charac- fenitrothion-degrading bacteria in soils from distant geographic terization of high acid tolerant and aluminum resistant yeasts areas. Microbes Environ 21:58–64 isolated from tea soils. Soil Sci Plant Nutr 51:671–674 Wakao N, Yasuda T, Jojima Y, Yamanaka S, Hiraishi A (2002) Enhanced Konishi S, Souta I, Takahashi J, Ohmoto M, Kaneko S (1994) Isolation growth of Acidocella facilis and related acidophilic bacteria at and characteristics of acid- and aluminum-tolerant bacterium. high concentrations of aluminum. Microbes Environ 17:98–104 Biosci Biotech Biochem 58:1960–1963 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct Kunito T, Shibata S, Matsumoto S, Oyaizu H (1997a) Zinc resistance sequencing of fungal ribosomal RNA genes for phylogenetics. In: of Methylobacterium species. Biosci Biotech Biochem 61:729– Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a 731 guide to methods and applications. Academic, London, pp 315–322

Journal

Annals of Microbiology – Springer Journals

Published: Nov 29, 2011

There are no references for this article.